The cloning of at least fifteen subtypes of P2 nucleotide receptors has presented a unique challenge to medicinal chemists: the design of selective agonists and antagonists for this multiplicity of receptors with few existing leads. These receptors regulate function of the central nervous system, epithelial cells, the immune system, the cardiovascular system, and smooth muscle. Our laboratory is developing selective agonists and antagonists for these receptors, for use both as pharmacological tools for probing receptor function and as potential therapeutic agents. P2X receptors are ligand-gated ion channels. P2Y receptors are G protein coupled receptors linked to the phosphatidyl inositol pathway as second messenger. The human P2Y1 receptor as representative of the P2Y family of metabotropic purine and pyrimidine nucleotide receptors may be modeled based on a rhodopsin template, and the resulting model is highly consistent with pharmacological and mutagenesis results. The entire P2Y receptor family has been modeled, and the clustering into two subfamilies with functionally conserved residues is evident. Charged residues in both the transmembrane and extracellular domains and two disulfide bridges essential for receptor activation have been identified. Selective P2Y1 receptor antagonists related to the adenine nucleotide MRS 2179 (N-methyl-2-deoxyadenosine-3,5-bisphosphate) developed in our lab and it carbocyclic analogues are under development. We have also synthesized nucleotides containing conformationally constrained ribose-like rings, in order to freeze a conformation that provides favorable affinity and/or selectivity at P2 receptors. As a result, we have identified the conformation preference of the P2Y1 receptor for the Northern ring conformation of the ribose. This conclusion applies to both agonists and antagonists. By freezing the ribose substitute in the receptor-preferred conformation, we have enhanced the potency of known agonists at the P2Y1 subtype by 200-300 fold. One ATP derivative containing a methylene carbon joining the second and third phosphate groups was qualitatively altered in its effects on the P2Y1 receptor: The ribose analogue is inactive, and the conformationally constrained analogue (Northern methanocarba) was a moderately potent agonist. The (N)-methanocarba nucleotide MRS2365 is a selective agonist for the P2Y1 receptor in comparison to all other P2Y subtypes, including the P2Y12 and P2Y13 receptors which are otherwise activated by the ribose equivalent. Thus, a constrained ring in the synthetic analogues designed and synthesized in our lab has greatly enhanced potency and selectivity for a P2Y subtype. In platelets, MRS2365 induces the characteristic shape change without progressing to aggregation. This compound promises to be an important tool in the study of platelet function. MRS 2500, a 2-iodoadenine-methanocarba bisphosphate nucelotide, in which the ribose-like ring is locked in the North conformation is the most potent known antaognist of the P2Y1 receptor. This antagonist has been shown to potently inhibit the ADP-induced aggregation of human platelets. This derivative has now been radiolabeled and shown to be useful as a receptor probe. This supports the view that both P2Y1 and P2Y12 receptor activation are needed for ADP-induced aggregation. These pharmacological tools will be useful in validating the possible use of P2Y1 antagonists as antithrombotic agents. Larger quantities of MRS2500 have now been synthesized, making it possible to study the effects in larger animal models. We are continuing the explore the structure activity relationships in this series of potent and selective P2Y1 receptor agonists and antagonists. The use of conformationally constrained nucleotides has also been extended to P2Y2, P2Y4, P2Y6, and P2Y11 subtypes. The Northern methanocarba ring system results in retention of high potency inagonists at all of the above subtypes, except P2Y6. The conformational requirements of the P2Y6 receptor are currently being explored in our section. Indications from molecular modeling and from novel analogues of UDP that the Southern (S) conformation of the ribose ring is favored at this subtype. This observation will allow the design and synthesis of more potent and selective agonists at this subtype. We recently completed the synthesis of the first enantiomerically pure (S) methanocarba nucleosides and nucleotides. Curiously, neither (N) or (S) isomers of UDP-glucose activate the P2Y14 receptor. We have explored the permissive SAR of the glucose region of UDP-glucose in recognition at the P2Y14 receptor. The exploration of newly synthesized analogues at the P2Y14 receptor has led us to identify novel selective agonist of nanomolar potency. Also, a relationship between this subtype and apoptosis, programmed cell death, has been discovered. Astrocytoma cells that express the P2Y6 receptor, when activated by UDP, are protected from apoptosis induced in control cells upon exposure to TNF, tumor necrosis factor. The protection involves activation of protein kinase C and subsequently the signaling kinase known as ERK. This may have relevance for degenerative and inflammatory conditions that involve TNF. Activation of P2Y6 receptors in mouse skeletal muscle, both in vitro and in vivo, has a protective effect. Also, we recently found that astrocytoma cells that express the P2Y12 receptor, when activated by ADP or more potent synthetic agonists, are protected from apoptosis induced in control cells upon exposure to TNF. This receptor occurs in the brain and the results suggest exploring the possible use of P2Y12 receptor activation in neuroprotection. Also, we recently reported that mouse pancreatic beta cells in culture that express the P2Y6 receptor, when activated by UDP or more potent synthetic agonists, are protected from apoptosis induced in control cells upon exposure to TNF. There is also a moderate increase of insulin secretion upon activation of the P2Y6 receptor. The results suggest exploring the possible use of P2Y6 receptor activation in treating diabetes. We are interested in desigining selective probes for other P2Y subtypes. We recently modeled the P2Y2 receptor (important for treatment of cystic fibrosis) and identified a highly potent and selective nucleotide agonist. MRS2698 is 300-fold selective for the human P2Y2 receptor in comparison to the human P2Y4 receptor. Activation of the P2Y2 subtype by UTP in cardiac myocytes is highly protective against ishcemic damage. In vivo studies to test this approach to cardioprotection are currently underway. The first antagonist of the P2Y13 receptor (MRS2211) has been developed. It is based structurally on pyridoxal-5-phosphate antagonists (such as PPADS), for which the SAR is being examined at all of the P2 receptor subtypes.